Assay for transformed alpha fetal protein

Abstract

A method for determining the existence of transformed alpha fetal protein in a sample. The first step is mixing said reagents by inversion before use. The second step is providing a microtiter plate with wells coated with synthetic TAFP diluted in a coating buffer, dispensing PSB buffer to wet the well surfaces, waiting a period of time and decanting the microtiter plate. The third step is providing a first washing of the well(s) with a wash buffer for removing unbound material. The fourth step is adding non-fat dry milk to the coating buffer and incubating the microtiter plate for a first period of time at room temperature for blocking nonspecific binding sites. The fifth step is providing a second washing of the well with a wash buffer for removing unbound material. The sixth step is adding anti-TAFP antibody diluted in a binding buffer for a second period of time for allowing the primary antipetide antibodies to bind. The seventh step is providing a third washing of the well(s) with a wash buffer for removing unbound material. The eighth step is adding diluted goat anti-rabbit immunoglobuin-horseradish peroxidase conjugate in TBST for binding of the primary to the secondary antibody and then incubating for a third period of time. The ninth step is providing a fourth washing of the well(s) with a TBST-containing buffer for removing unbound material. The tenth step is adding horseradish peroxidase substrate and incubating at room temperature for developing color. The eleventh step is adding stop solution to said well after a fourth period of time for stopping the test. Finally, the twelfth step is determining the absorbance of the alpha fetal protein peptide at a wavelength using a microplate reader.

What is claimed is:  
     
         1 . A method for determining the existence of transformed alpha fetal protein in a sample, said method comprising the steps of: 
 a) providing a microtiter plate with wells coated with synthetic TAFP diluted in a coating buffer, dispensing PSB buffer to wet said well surfaces, waiting a period of time and decanting said plate;    b) washing said well with a wash buffer for removing unbound material;    c) adding non-fat dry milk to said coating buffer and incubating said microtiter plate for a first period of time;    d) washing said well with a wash buffer for removing unbound material;    e) adding anti-TAFP antibody diluted in a binding buffer for a second period of time for allowing the primary antipetide antibodies to bind;    f) washing said well with a wash buffer for removing unbound material;    g) adding diluted goat anti-rabbit immunoglobuin-horseradish peroxidase conjugate in TBST for binding of the primary to the secondary antibody and then incubating for a third period of time;    h) washing said well with a TBST-containing buffer for removing unbound material;    i) adding horseradish peroxidase substrate and incubating to develop color;    j) adding stop solution to said well for stopping the test; and    k) determining the absorbance at a selected wavelength.    
     
     
         2 . The method of  claim 1 , wherein said wavelength is approximately 410 nm.  
     
     
         3 . An apparatus for determining the existence transformed alpha fetal protein comprising: 
 an anti-TAFP coated plate;    color-coded reagents for determining test standards;    test controls;    an assay buffer;    a wash concentrate;    a horseradish peroxidase substrate; and,    a stop solution for stopping the test.